This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Glycoconjugates and oligosaccharides, molecules containing specific carbohydrate structures, constitute a major class of milk components that inhibit pathogens. We are particularly interested in glycosphingolipids, which consist of an oligosaccharide sphingosine base with an N-linked fatty acyl group. Recent discoveries have indicated that sulfatides [unreadable]glycosphingolipids carrying a sulfate ester group on the glycan [unreadable]may play an essential role in fighting HIV. However, only sulfatides from human milk, not those from other mammalian sources, e.g., bovine brain, have this bio-activity. We have developed approach to extract glycosphingolipids and glycosaminoglycans out of human milk and study their structures by LesTQ Orbitrap MS. The pooled human breast milk samples were lyophilized, reconstituted and put through Folch partition. The fractions were dried and extracted in turn and separated with a silicic acid column. Sulfatide standards were used as a control. Structural characterization was based on tandem MS analyses performed on an LTQ-Orbitrap mass spectrometer coupled with a Triversa Nanomate system in the negative ion mode. Under the designed work flow, CID and HCD were applied as collision methods to the precursor ion which had the highest signal intensity. From the negative-ion ESI MS spectra obtained with the LTQ-Orbitrap, numerous acidic glycosphingolipids were detected. HCD was used for observing the lower mass range fragments, while CID was applied to compensate for the lower efficiency of HCD in the higher mass range. By combining these techniques, we were able to assign both the glycan moiety and the ceramide structures across the range of low and high abundance peaks. The fatty acyl chain lengths varied from 16 to 24 backbone carbons with different degrees of unsaturation. Since the exact masses of GSLs with sulfate or phosphate groups are very close, it was impossible to isolate ions having a single composition prior to fragmentation, but diagnostic product ions were present in the MS2 spectra. The glycosphingolipid extraction method we optimized reduced content of phospholipids and facilitated observation of the sulfated GSLs. By this LTQ Orbitrap MS work flow, we identified hundreds of lipids in the human milk anti-HIV fraction. Ongoing experiments should reveal further compound structures in the human milk GSL and GAG fractions and test the activity of synthetic sulfatides against HIV.